Patent application title: Process for oxidizing unfractionated heparins and detecting presence or absence of glycoserine in heparin and heparin products

Abstract:

The invention provides a process for preparing heparin products with a
reduced content of glycoserine. A method for detecting glycoserine in
preparations of heparin is also provided.

Claims:

1. A process for preparing at least one decolorized heparin product,
exclusive of commercially available LMWHs regulated by the USFDA as of
the filing date of this application, from heparin comprising:a.
purification of the heparin by oxidation with about 4% to about 10% by
weight relative to the heparin of at least one permanganate salt chosen
from potassium permanganate, sodium permanganate, and quaternary ammonium
permanganate, wherein oxidation occurs at a temperature ranging from
approximately 35.degree. C. to approximately 90.degree. C.; andb.
depolymerization of the oxidized heparin to obtain said at least one
decolorized heparin product.

2. The process according to claim 1, wherein the heparin product is
purified after depolymerization.

3. The process according to claim 1, wherein the concentration of the at
least one permanganate is about 8%.

4. The process according to claim 1, wherein the at least one permanganate
is potassium permanganate.

5. The process according to claim 1, wherein the heparin product is a low
molecular weight heparin which is not enoxaparin or enoxaparin sodium.

6. The process according to claim 1, wherein the heparin product is an
ultralow molecular weight heparin.

7. The process according to claim 1, wherein oxidation occurs at a
temperature ranging from approximately 40.degree. C. to approximately
80.degree. C.

8. The process according to claim 1, wherein said at least one heparin
product is glycoserine-free.

9. The process according to claim 1, wherein said at least one heparin
product has a reduced glycoserine content.

10. A method for determining the glycoserine content of a sample of a
heparin or a heparin product comprising:a. treating the sample; andb.
analyzing the sample using a chromatography process to detect the
presence or absence of glycoserine and/or oxidized glycoserine residues
in the sample.

11-38. (canceled)

39. A substantially pure compound having the formula:

40. A substantially pure compound having the formula:

41. A substantially pure compound having the formula:

42. A process for preparing at least one decolorized heparin product
chosen from fraxiparin, fragmin, innohep (logiparin), normiflo, embollex
(sandoparin), fluxum (mimidalton), clivarine, and hibor from heparin
comprising:a. purification of the heparin by oxidation with about 4% to
about 10% by weight relative to the heparin of at least one permanganate
salt chosen from potassium permanganate, sodium permanganate, and
quaternary ammonium permanganate, wherein oxidation occurs at a
temperature ranging from approximately 35.degree. C. to approximately
90.degree. C.; andb. depolymerization by a manufacturer of the oxidized
heparin according to a process to obtain said decolorized heparin
product.

43. A process for preparing at least one decolorized heparin product
chosen from fraxiparin, fragmin, innohep (logiparin), normiflo, embollex
(sandoparin), fluxum (mimidalton), clivarine, and hibor
comprising:depolymerizing heparin oxidized with about 4% to about 10% by
weight relative to the heparin of at least one permanganate salt chosen
from potassium permanganate, sodium permanganate, and quaternary ammonium
permanganate, wherein oxidation occurs at a temperature ranging from
approximately 35.degree. C. to approximately 90.degree. C., according to
a process to obtain said decolorized heparin product.

44-45. (canceled)

46. A process for preparing decolorized enoxaparin from heparin
comprising:a. purification of the heparin by oxidation with about 4% to
about 10% by weight relative to the heparin of at least one permanganate
salt chosen from potassium permanganate, sodium permanganate, and
quaternary ammonium permanganate, wherein oxidation occurs at a
temperature ranging from approximately 35.degree. C. to approximately
90.degree. C.; andb. depolymerization by a manufacturer other than one
chosen from Aventis Pharma SA, its fully owned subsidiaries, and its
successors and assigns, and agents of Aventis Pharma SA, its fully owned
subsidiaries, and its successors and assigns, of the oxidized heparin
according to a process to obtain said enoxaparin.

47. A process for preparing decolorized enoxaparin comprising:
depolymerization according to a process by a manufacturer other than one
chosen from Aventis Pharma SA, its fully owned subsidiaries, and its
successors and assigns, and agents of Aventis Pharma SA, its fully owned
subsidiaries, and its successors and assigns, of heparin oxidized by
about 4% to about 10% by weight relative to the heparin of at least one
permanganate salt chosen from potassium permanganate, sodium
permanganate, and quaternary ammonium permanganate, wherein oxidation
occurs at a temperature ranging from approximately 35.degree. C. to
approximately 90.degree. C.;, to obtain said enoxaparin.

48-70. (canceled)

Description:

[0001]This application claims the benefit of French Patent Application No.
______ entitled "Process for Oxidizing Unfractionated Heparins and
Detecting Presence or Absence of Glycoserine in Heparin and Heparin
Products," which was filed Mar. 24, 2004, and which is incorporated
herein in its entirety.

[0002]One embodiment of the present invention relates to a process for
oxidizing crude heparin preparations using at least one permanganate salt
chosen from potassium permanganate, sodium permanganate, and quaternary
ammonium permanganate. The resulting heparin preparations are relatively
free of glycoserine residues and are useful, for example, for preparing
low molecular weight heparins (LMWHs) and ultra-low molecular weight
heparins (ULMWHs) that are relatively free of glycoserine residues.
Another embodiment of the invention relates to a method for detecting the
presence of glycoserine and glycoserine derivatives in preparations of
heparin and of fragmented heparin.

[0003]Heparins are biologically active members of the glycosaminoglycan
family and can be extracted from, e.g., bovine and porcine sources.
Heparins have anticoagulant and antithrombotic properties that make them
useful in treatment of thromboses, such as arterial and venous
thromboses. As isolated, natural heparin molecules have a high
anticoagulant activity, which can lead to hemorrhaging. Moreover, heparin
molecules are sensitive to particular serum factors and, consequently,
must be administered in large doses to provide antithrombotic benefits
greatly increasing the risk of hemorrhaging.

[0005]Each LMWH manufacturer of an approved product utilizes a distinct
process of depolymerization. Unless two manufacturers use the same
process, this process distinctness results in LMWHs with distinct
chemical structures and, therefore, differing pharmacological activity
and different approved indications for clinical use.

[0006]Therefore, LMWHs are structurally differentiated by the
depolymerization processes used for their manufacture (R. J. Linhardt, et
al, Seminars in Thombosis and Hemostatis 1999; 25(3 Supp.): 5-16). As a
result, LMWHs are more heterogeneous than heparin. Each different process
causes unique and highly complex structural modifications to the
polysaccharide chains. These modifications include differences in chain
lengths and chain sequences, as well as structural fingerprints.
Consequently, the different commercial LMWHs each have distinctive
pharmacological profiles and different approved clinical indications.

[0007]During the process for preparing enoxaparin sodium, sold under the
tradename Lovenox® in the US and Clexane® in some other
countries, from pure heparin, the aqueous-phase alkaline depolymerization
process produces a partial but characteristic conversion of the
glucosamines of the reducing ends of the oligosaccharide chains.

[0008]The first step of this conversion consists of a glucosamine
⇄ mannosamine epimerization (T. Toida, et al., J.
Carbohydrate Chemistry, 15(3), 351-360 (1996)); the second step is a
6-O-desulfation of the glucosamine, leading to the formation of
derivatives called "1,6 anhydro" (International patent application WO
01/29055).

[0010]The percentage of oligosaccharide chains whose end is modified with
a 1,6-anhydro bond is a structural characteristic of the oligosaccharide
mixture of Lovenox® (enoxaparin sodium). Based on current knowledge,
between 15% and 25% of the components of Lovenox® (enoxaparin sodium)
have a 1,6-anhydro structure at the reducing end of their chain.

[0011]Recently, new processes for the preparation of heparin fragments
employing depolymerization in the presence of a strong base have yielded
ultra-low molecular weight heparins having a weight average molecular
weight ranging from approximately 1500 to approximately 3000 Daltons
(ULMWHS) as described, e.g., in U.S. Published Patent Application No.
2002-0055621 A1, specifically incorporated by reference herein.

[0012]Compositions of LMWH and ULMWH fragments are heterogeneous and
contain individual heparin fragments of varying lengths and molecular
weights.

[0013]Both heparin itself and heparin fragment mixtures have limited shelf
lives, at least in part, because they become colored during storage. Once
they develop a color, those compositions may not be commercially
desirable for injection into patients. A process that yields heparin that
resists coloration is therefore highly desirable for commercial reasons.
Such coloration-resistant heparin can, of course, be used to prepare
coloration-resistant LMWHs and/or ULMWHs by methods known to those
skilled in the art. Moreover, a method for assessing the potential of
heparin preparations to colorize would be useful in the manufacture of
heparin, LMWH, and ULMWH.

[0014]It is accordingly an embodiment of the invention to provide a
process for preparing heparin that is resistant to coloration. The
resulting heparin may subsequently be employed to produce LMWH and ULMWH
mixtures, particularly commercially available mixtures, such as
fraxiparin, fragmin, innohep (or logiparin), normiflo, embollex (or
sandoparin), fluxum (or mimidalton), clivarine, and hibor, that are
resistant to coloration. As will be evident herein, certain embodiments
of the invention can utilize enoxaparin, sold commercially as
Lovenox® (enoxaparin sodium) in the US and Clexane® (enoxaparin
sodium) in some other countries. Enoxaparin is commercially available
from Aventis Pharma S.A. and Aventis Pharmaceuticals, Inc. Other
embodiments do not utilize enoxaparin. Another embodiment of the
invention is to provide a method for monitoring the tendency of heparin
and mixtures of heparin fragments to color.

[0015]One embodiment of the invention provides a novel process for
preparing heparin using at least one permanganate salt chosen from
potassium permanganate, sodium permanganate, and quaternary ammonium
permanganate, such as potassium permanganate, in a heparin oxidation
process to produce glycoserine-free heparinic compositions. That process
is described below. The applicant has discovered that if at least one of
those permanganates salts, such as potassium permanganate, is utilized in
the oxidation step, glycoserine-free or low glycoserine heparin is
obtained. It was also discovered that removing the glycoserine residues
and/or glycoserine derivatives from heparin can lead to products with
improved commercial characteristics. Those reduced-glycoserine products
are colorless or nearly colorless and show a decreased tendency to
colorize relative to products made with heparin that has not been
oxidized in the presence of potassium permanganate.

[0016]Additionally, at least one embodiment of the present invention
includes a method for detecting glycoserine residues (or the lack
thereof) in heparin compositions. This method includes pre-treatment of
the heparin with one or more heparinases and detection of the acetylated
groups using chromatography. Further detailed descriptions of the present
invention are provided below.

[0017]In accordance with the invention, conditions are provided that
permit the chemoselective elimination of glycoserine residues and
glycoserine derivatives, such as oxidized glycoserines, from heparin.

[0018]In one embodiment of the invention, at least one permanganate salt
chosen from potassium permanganate, sodium permanganate, and quaternary
ammonium permanganate, such as potassium permanganate is used in a
heparin oxidation process comprising oxidation with about 4% to about 10%
by weight relative to the heparin of said at least one permanganate salt
so as to obtain a low glycoserine or glycoserine-free heparin. If the
resulting low glycoserine or glycoserine-free heparin is depolymerized
using methods known in the art, low glycoserine or glycoserine-free LMWHs
or ULMWHs such as, fraxiparin, fragmin, innohep (or logiparin), normiflo,
embollex (or sandoparin), fluxum (or mimidalton), clivarine, and hibor
may be obtained, but not enoxaparin sodium.

[0019]Commercial processes, however, for manufacturing those commercial
products are believed to contain proprietary details that, at least in
the case of enoxaparin, can affect the biological properties of the final
product, as explained in Feb. 19, 2003, Citizen Petition and Citizen
Petition Supplement (03P-0064/CP1) filed on behalf of Aventis
Pharmaceuticals Inc., a subsidiary of Aventis SA, the assignee and
publicly available from the United States Food and Drug Administration
(USFDA).

[0020]Hence an embodiment of the invention relates to a process for
preparing at least one decolorized heparin product chosen from
fraxiparin, fragmin, innohep (logiparin), normiflo, embollex
(sandoparin), fluxum (minidalton), clivarine, and hibor from heparin
comprising:

[0021]a) purification of the heparin by oxidation with about 4% to about
10% by weight relative to the heparin of at least one permanganate salt
chosen from potassium permanganate, sodium permanganate, and quaternary
ammonium permanganate, wherein oxidation occurs at a temperature ranging
from approximately 35° C. to approximately 90° C.; and

[0022]b) depolymerization by a manufacturer of the oxidized heparin
according to a process to obtain said heparin product.

[0023]Alternatively, the manufacturer can depolymerize heparin oxidized
with about 4% to about 10% by weight relative to the heparin of at least
one permanganate salt chosen from potassium permanganate, sodium
permanganate, and quaternary ammonium permanganate, wherein oxidation
occurs at a temperature ranging from approximately 35° C. to
approximately 90° C. according to a process to obtain said heparin
product Regarding enablement, a manufacturer of one of the commercial
products recited in this aspect of the invention will be able to practice
these embodiments of the invention.

[0025]Another subject of the invention is the use of at least one of the
permanganate salt, such as potassium permanganate, in the heparin
oxidation process described above, i.e., oxidation with about 4% to about
10% by weight relative of said to the heparin of said at least one of the
permanganate salts to obtain a heparin that is decolorized. If the
resulting decolorized heparin is depolymerized, decolorized heparin
products including LMWHs or ULMWHs such as fraxiparin, fragmin, innohep
(or logiparin), normiflo, embollex (or sandoparin), fluxum (or
minidalton), clivarine, and hibor might be obtained, subject to the
comment on commercial processes made above.

[0026]Another subject of the invention is the use of potassium
permanganate in the heparin oxidation process described above to obtain a
heparin that is low glycoserine or glycoserine-free and decolorized. If
the resulting low glycoserine or glycoserine-free and decolorized heparin
is depolymerized, decolorized heparin products including LMWHs or ULMWHs
such as fraxiparin, fragmin, innohep (or logiparin), normiflo, embollex
(or sandoparin), fluxum (or minidalton), clivarine, and hibor might be
obtained, as explained above.

[0027]In the present invention, the phrase "heparin" means all forms of
heparin other than a heparin product, including without limitation crude
heparin, upgraded heparin and purified heparin.

[0028]In the present invention, the phrase "upgraded heparin" means
heparin having an increased anti Xa activity compared to the starting
heparin prior to upgrading. For example, and without limitation, if the
anti Xa activity of the starting heparin is 140 IU/mg, the upgraded
heparin may have an anti Xa activity of 150 IU/mg or 200 IU/mg.

[0029]In the present invention, the phrase "low molecular weight heparin"
or "LMWH" means a mixture of polysaccharides obtained from heparin and
having a weight average molecular weight greater than approximately 3,000
Daltons and less than approximately 10,000 Daltons.

[0030]In the present invention, the phrase "ultra-low molecular weight
heparin" or "ULMWH" means a mixture of polysaccharides obtained from
heparin having a weight average molecular weight ranging from
approximately 1500 to approximately 3000 Daltons.

[0031]In the present invention, the phrase "depolymerization" (and
variations thereof such as depolymerize or depolymerizing) includes all
types of depolymerization, including without limitation fragmentation,
chemical and enzymatic depolymerization, and other methods of preparing
fragments of heparin, including without limitation fractionation.

[0032]In the present invention, the phrase "low glycoserine" means a
glycoserine (such as, for example, as illustrated below at paragraph 63)
percent less than or equal to 0.3% of all disaccharide residues in the
sample have a glycoserine attached.

[0033]In the present invention, the phrase "glycoserine-free" means a
glycoserine (such as, for example, as illustrated below at paragraph 63)
percent less than or equal to 0.1% of all disaccharide residues in the
sample have a glycoserine attached.

[0034]In the present invention, the phrase "relatively glycoserine-free"
means a glycoserine (such as, for example, as illustrated below at
paragraph 63) percent less than or equal to 0.3% of all disaccharide
residues in the sample have a glycoserine attached.

[0035]In the present invention, the phrase "decolorized" means less than
or equal to 0.2 absorbance units in an accelerated stability test such as
that described below under the heading "Preparation for colorimetric
analysis of the sample." FIG. 1 reflects the results of an accelerated
stability test for enoxaparin sodium.

[0036]In the present invention, the phrase "coloration" means greater than
0.2 absorbance units in an accelerated stability test such as that
described below under the heading "Preparation for calorimetric analysis
of the sample." FIG. 1 reflects the results of an accelerated stability
test for enoxaparin sodium.

[0037]In the present invention, the phrase "coloration-resistant" means
less than or equal to 0.2 absorbance units in an accelerated stability
test such as that described below under the heading "Preparation for
calorimetric analysis of the sample." FIG. 1 reflects the results of an
accelerated stability test for enoxaparin sodium.

[0039]In one embodiment of the process of the invention, 4 to 10% by
weight relative to the heparin of at least one permanganate chosen from
potassium permanganate, sodium permanganate, and quaternary ammonium
permanganate, such as potassium permanganate, is used for preparing
heparins purified by oxidation. In a related embodiment, approximately 8%
by weight relative to the heparin of potassium permanganate is used in a
process for preparing heparins purified by oxidation.

[0040]In another embodiment of the invention, the process for preparing
heparins purified by oxidation with at least one permanganate salt chosen
from potassium permanganate, sodium permanganate, and quaternary ammonium
permanganate, such as potassium permanganate, is performed at a
temperature ranging from approximately 35° C. to approximately
90° C. In a related embodiment, the process for preparing heparins
purified by oxidation with potassium permanganate is performed at a
temperature from approximately 40° C. to approximately 80°
C.

[0041]An additional subject of the invention is at least one composition
chosen from low glycoserine, glycoserine-free, and decolorized LMWHs and
ULMWHs, exclusive of products approved by the USFDA as of the filing date
of this application, which can be obtained according to a process
comprising the following steps:

[0042]a) purification of the heparin by the action of 4 to 10% by weight
relative to the heparin of at least one permanganate salt chosen from
potassium permanganate, sodium permanganate, and quaternary ammonium
permanganate, such as potassium permanganate, wherein oxidation occurs at
a temperature ranging from approximately 35° C. to approximately
90° C.;

[0043]b) depolymerization of the heparin; and

[0044]c) optionally, purification of the at least one composition.

[0045]An additional subject of the invention is at least one composition
chosen from low glycoserine, glycoserine-free, and decolorized LMWHs and
ULMWHs, exclusive of products approved by the USFDA as of the filing date
of this application, which can be obtained according to a process
comprising depolymerizing heparin oxidized by the action of 4 to 10% by
weight relative to the heparin of at least one permanganate salt chosen
from potassium permanganate, sodium permanganate, and quaternary ammonium
permanganate, wherein oxidation occurs at a temperature ranging from
approximately 35° C. to approximately 90° C.

[0046]An additional subject of the invention is a method for determining
the glycoserine content of a sample of a heparin or a heparin product,
including, but not limited to, LMWH and ULMWH and commercially available
forms thereof, comprising:

[0047]a) treating a sample chosen from heparins and heparin products; and

[0048]b) thereafter, using a chromatography process for determining the
presence of glycoserine residues in the sample.

[0049]In one embodiment of the invention, the sample chosen from heparins
and heparin products is treated by depolymerization through the action of
a heparinase before it is analyzed in the chromatography process. In
another embodiment, the sample chosen from heparins and heparin products
is depolymerized by the action of a mixture of heparinases. For instance,
the mixture of heparinases may comprise heparinase 1 (EC 4.2.2.7.),
heparinase 2 (heparin lyase II), and heparinase 3 (EC 4.2.2.8.).

[0050]In another embodiment of the invention, anion-exchange
chromatography (SAX--Strong Anionic Exchange) is used for determining the
presence (or absence) of glycoserine in the sample after the sample is
treated. In a related embodiment of the invention, the stationary phase
for anion-exchange chromatography is grafted with quaternary ammonium
derivatives, including, for example, --NMe3+. As used herein, the
term "strong anion exchange chromatography" (SAX) encompasses anion
exchange chromatography conducted on any resin that maintains a constant
net positive charge in the range of about pH 2-12. In certain embodiments
of the invention, strong anion exchange chromatography uses a solid
support functionalized with quaternary ammonium exchange groups. For
example, columns such as Spherisorb® SAX (Waters Corp, Milford Mass.)
may be used having particle size of about 5 μm, a column length of
about 25 cm and a column diameter of between about 1 mm and about 4.6 mm
may be used. In another embodiment, CTA-SAX chromatography may be used,
as described in the U.S. patent application entitled "Method for
Determining Specific Groups Constituting Heparins or Low Molecular Weight
Heparins" filed with the USPTO on even date herewith and hereby
incorporated herein by reference solely for references to CTA-SAX
chromatography. CTA-SAX chromatography is defined in said application as
anion exchange chromatography conducted on a quaternary ammonium salt
dynamically coated on a reversed phase silica column that maintains a
constant net positive charge in the range of about pH 2 to about pH 12.

[0051]An embodiment of the present invention thus provide a method for
quantifying the glycoserine content of a sample of a heparin or heparin
product by analyzing the sample for glycoserine content. In one
embodiment, analysis of the sample includes enzymatically digesting the
sample and quantifying the content of the glycoserine residues in the
digested sample by chromatography methods such as HPLC. In a related
embodiment, CTA-SAX or SAX may be utilized to quantify the content of
glycoserine residues. For example, and without limitation, the
glycoserine content of the sample may be reduced below 2.0% of the
sample, below 0.3% of the sample, or below 0.1% of the sample. The
glycoserine content of the sample may also be 0.0% (i.e., undetectable).

[0052]In one embodiment of the method for determining the glycoserine
content of a sample of a heparin or a heparin product, the mobile phase
for the chromatography step is transparent to ultraviolet (UV) light in a
range from about 200 nm to about 400 nm. The mobile phase may comprise,
for example, sodium perchlorate, methanesulfonate salts, or phosphate
salts.

[0053]In one embodiment of the method for determining the glycoserine
post-treatment content of a sample chosen from heparins and heparin
products, polysaccharides are detected by their UV absorption following
chromatography. The UV absorption of the polysaccharides may be measured
at two wavelengths for example, 202 nm and 240 nm, following
chromatography so that the absorption signals of non-acetylated
polysaccharides cancel out.

[0054]Another embodiment of the method provides a method for monitoring
the glycoserine content of a sample of a heparin or a heparin product
comprising:

[0058]a) purification of the heparin by oxidation with about 4% to about
10% by weight relative to the heparin of at least one permanganate salt
chosen from potassium permanganate, sodium permanganate, and quaternary
ammonium permanganate, wherein oxidation occurs at a temperature ranging
from approximately 35° C. to approximately 90° C.; and

[0059]b) depolymerization of the oxidized heparin.

[0060]In an additional embodiment of the invention, the glycoserine
content of a sample chosen from heparins and heparin products may be
quantified, for example, either by external calibration or internal
calibration. Internal calibration can comprise the use of an internal
standard such as 2-naphthol-3,6-disulfonic acid. In another related
embodiment, the solution to be assayed may contain about 0.15 g/l of
2-naphthol-3,6-disulfonic acid.

[0061]Another embodiment of the method provides a method of predicting the
tendency of a heparin or a heparin product to colorize, comprising
measuring the glycoserine content of said heparin or heparin product. In
a related embodiment, the glycoserine content is quantified by external
or internal calibration. The internal calibration may comprise the use of
an internal standard. In a related embodiment, the internal standard is
2-naphthol-3,6-disulfonic acid or about 0.15 g/l of 2-naphthol-3,6
disulfonic acid.

[0062]In one embodiment of the invention, the chromatography step detects
the tetrasaccharide of the glycoserine-linking domain of heparin
molecules. The tetrasaccharide of the glycoserine-linking domain of
heparin is:

[0063]One skilled in the art will appreciate that this method and the
aspects described herein may be used to determine the glycoserine content
of LMWHs and ULMWHs known in the art, as well as those available
commercially. Additional advantages of the invention are set forth in
part in the description that follows, and in part will be obvious from
the description, or discernible to one of ordinary skill practicing the
invention. The advantages of the invention will be realized and attained
by means of the elements and combinations particularly pointed out in the
appended claims.

[0064]It is to be understood that both the foregoing general description
and the following detailed description are exemplary and explanatory only
and are not restrictive of the invention, as claimed.

[0065]Likewise, the accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several embodiments
of the invention and together with the description, serve to explain the
principles of the invention.

[0070]Reference will now be made in detail to exemplary embodiments of the
invention, which are illustrated in the accompanying drawings.

[0071]The presence of glycoserine residues in preparations of heparin,
LMWH, and ULMWH may cause quality and stability problems that decrease
the commercial value of the product. For example, in enoxaparin stability
tests, glycoserine residues increase the rate of coloration and may lead
to product batches that are not within approved manufacturing
specifications. See FIG. 1. Controlling the amount of glycoserine in
heparin makes it possible to better standardize the resulting commercial
products, including LMWHs, and ULMWHs. As a consequence, the risk of
producing product batches that cannot be sold may be decreased.

[0072]It has been demonstrated that the action of at least one of the
permanganate salts mentioned herein, such as potassium permanganate,
makes it possible to selectively cleave the glycoserine residue in the
heparin chain. Moreover, the sites of action of permanganate, the
mechanism of action of permanganate, and the structures resulting from
permanganate action have been characterized.

[0073]Consequently, a subject of the invention is glycoserine-free,
decolorized heparin and a process for preparing glycoserine-free,
decolorized heparin, comprising the following steps:

[0074]a) treatment of the heparin with from about 4% to about 10% relative
to the weight of the heparin of at least one permanganate chosen from
potassium permanganate, sodium permanganate, and quaternary ammonium
permanganate, such as potassium permanganate, at a temperature ranging
from approximately 35° C. to approximately 90° C. according
to the process as described above; and

[0075]b) purification of the glycoserine-free, decolorized heparin.

[0076]In one embodiment, the temperature ranges from about 40° C.
to about 80° C. In this range, 8% relative to the weight of the
heparin of permanganate treatment maintains its effectiveness in
eliminating glycoserine residues. And at permanganate concentrations
equal to or greater than 4% relative to the weight of the heparin of
permanganate at a temperature of approximately 80° C., treatment
according to the invention virtually eliminates detectable glycoserine
residues. Below 4% relative to the weight of the heparin of permanganate,
the treatment may no longer be sufficient to eliminate glycoserine
residues (as seen in example 9 below). In fact, the concentration of
potassium permanganate used in the oxidation step is more important than
temperature in reducing or eliminating glycoserine residues.

[0077]Heparin prepared according to the invention may in turn be used to
prepare other glycoserine-free and decolorized heparin products, for
example, fraxiparin, enoxaparin, fragmin, innohep (or logiparin),
normiflo, embollex (or sandoparin), fluxum (or mimidalton), clivarine and
hibor.

[0078]Another embodiment of the invention is directed to a process for
preparing decolorized enoxaparin from heparin comprising:

[0079]a) purification of the heparin by oxidation with about 4% to about
10% by weight relative to the heparin of at least one permanganate salt
chosen from potassium permanganate, sodium permanganate, and quaternary
ammonium permanganate, wherein oxidation occurs at a temperature ranging
from approximately 35° C. to approximately 90° C.; and

b) depolymerization by a manufacturer other than one chosen from Aventis
Pharma SA, its fully owned subsidiaries, and its successors and assigns,
and agents of Aventis Pharma SA, its fully owned subsidiaries, and its
successors and assigns, of the oxidized heparin according to a process to
obtain said enoxaparin.

[0080]Additionally, another embodiment of the invention is directed to a
process for preparing decolorized enoxaparin comprising:

[0081]depolymerization according to a process by a manufacturer other than
one chosen from an Aventis company, its successors, and assigns, and
agents of an Aventis company, its successors, and assigns, of heparin
oxidized by about 4% to about 10% by weight relative to the heparin of at
least one permanganate salt chosen from potassium permanganate, sodium
permanganate, and quaternary ammonium permanganate, wherein oxidation
occurs at a temperature ranging from approximately 35° C. to
approximately 90° C., to obtain said enoxaparin.

[0083]Therefore, an additional subject of the invention is a
glycoserine-free, decolorized LMWH and a process for preparing
glycoserine-free, decolorized LMWH, exclusive of available LMWHs
regulated by the USFDA as of the filing date of this application,
comprising the following steps:

[0084]a) treatment of the heparin with from about 4% to about 10% relative
to the weight of the heparin of at least one permanganate chosen from
potassium permanganate, sodium permanganate, and quaternary ammonium
permanganate, such as potassium permanganate, at a temperature ranging
from approximately 35° C. to approximately 90° C. according
to the process as described above;

[0085]b) purification of the glycoserine-free, decolorized heparin; and

[0086]c) depolymerization of the glycoserine-free, decolorized heparin to
produce said glycoserine-free, decolorized LMWH

[0087]The structure below shows the permanganate cleavage points in
heparin, demonstrating the mechanism of action of potassium permanganate
during oxidation of the heparin molecules:

[0088]Without being limited to theory, it is believed that when heparin is
treated according to the method of the invention, the permanganate salt
acts on the vicinal diols of glucuronic acid, making it possible to
selectively eliminate the serine residues. Although potassium
permanganate is much more selective, sodium periodate also cleaves
heparin at these sites (See H. E. Conrad, Heparin-Binding Proteins, 130,
Academic Press (1998)). However, in contrast to reactions with
permanganate, the reaction of sodium periodate with heparin degrades the
ATIII site and results in an undesirable loss of anticoagulant activity.
Id.

[0089]Treatment with potassium permanganate according to the invention
also degrades the glycoserine amino acid to an acid, thereby eliminating
the source of a component required for the heparin coloration reactions
(Maillard reactions). The action of permanganate on heparin according to
a method of the invention can be illustrated by the following reactions,
which are not exhaustive:

[0090]To identify the cleavage points, the general method is that heparin
treated with, for example, potassium permanganate according to the
invention is subjected to the action of heparinase III. This enzyme is
highly specific for the non-sulfated regions of heparin. It selectively
depolymerizes the disaccharide units containing uronic acid free of
2-O-sulfate and the domain for heparin binding to the protein (the
protein that carries the heparin strands is a serine-glycine chain).
Formation of the ΔIVa, ΔIVs disaccharides of the
ΔIVa-Gal-Gal-Xyl-Ser component or of any oxidized derivative of
this--linking region carrying the IVa disaccharide is mainly observed
following digestion with heparinase III. This enzyme does not affect the
remainder of the heparin chain. Consequently, the depolymerized material
contains a mixture of heparin oligosaccharides (disaccharides to
tetrasaccharides) and other polysaccharides. This mixture requires a
pre-treatment to eliminate the heparin chains before it can be analyzed
by High Performance Liquid Chromatography (HPLC). One skilled in the art
will be familiar with a number of methods for eliminating heparin chains,
including, for example, ultracentrifugation through a Millipore membrane
(5 kDa) or methanol precipitation followed by centrifugation. The
solution thus prepared can then be analyzed by HPLC. The following
fragments can be identified by HPLC (porous graphite)/mass spectrometry
coupling ("MM" means molecular mass):

[0091]The structures of the tetrasaccharide MM=690 and of the
trisaccharide MM=588 were confirmed by Nuclear Magnetic Resonance (NMR).

[0092]Those fragments demonstrate that treatment with potassium
permanganate according to the invention selectively acts on the region of
protein-linking and eliminates the serine residue. The compound MM=690 is
the result of reaction 1 followed by enzyme digestion. The compound
MM=511 is the result of reaction 2 followed by enzyme digestion. The
compound MM=588 is the result of reaction 3 followed by enzyme digestion.

[0093]The foregoing compounds MM=511, MM=588 and MM=690 may be isolated
and obtained in substantially pure form. As used herein, "substantially
pure" means sufficiently pure to identify the compounds by mass
spectroscopy or NMR. In one embodiment, a substantially pure compound is
one which is at least 80% pure. In another embodiment, a substantially
pure compound is one which is at least 90% pure. In an aspect of the
invention, a method is provided for determining the oligosaccharide
content of a sample of a heparin or heparin product comprising
depolymerizing the sample and analyzing the sample using a chromatography
process to detect oligosaccharides chosen from MM=511, MM=588 and MM=690.
In a related embodiment, the oligosachharide content is quantified by
external or internal calibration. In another embodiment, the internal
calibration comprises and internal standard. In a related embodiment, the
internal standard is a substantially pure compound selected from MM=511,
MM=588 and MM=690.

[0094]For structural identification, utilizing HPLC, the tetrasaccharide
characteristic of the binding linking domain (glycoserine) is isolated
and identified by NMR (for example, a crude heparin can be selectively
depolymerized by the action of heparinase III):

[0096]Because coloration problems potentially impact the shelf-life of
heparin, LMWH, and ULMWH preparations, and therefore require quality
control during the manufacturing process, an additional subject of the
invention is a method for analyzing such preparations in order to detect
and/or quantify the presence of glycoserine and its oxidized derivatives.
An embodiment of the method comprises the following steps:

[0097]a) depolymerization of the sample;

[0098]b) whether the sample is heparin or a heparin product, separation of
the resulting oligosaccharides by HPLC; and

[0100]In one embodiment of the invention, depolymerization of the sample
may be achieved through the action of a mixture of heparinases
comprising, for example, heparinase 1 (EC 4.2.2.7.), heparinase 2
(heparin lyase II), and heparinase 3 (EC 4.2.2.8.), which are available
from Grampian Enzymes. The enzymatic depolymerization may be carried out
over a period of hours, which can be determined by one skilled in the
art, at an appropriate temperature, which can also be determined by one
skilled in the art. For example, the enzymatic depolymerization may be
carried out for 48 hours at ambient temperature by mixing 20 μl of an
aqueous solution containing 20 mg/ml of the heparin to be assayed, and
100 μl of a 100 mM acetic acid/NaOH solution, at pH 7.0, containing 2
mM of calcium acetate and 2 mg/ml of BSA, with 20 μl of a stock
solution of comprising a mixture of heparinase 1, heparinase 2, and
heparinase 3, comprising 0.5 units/ml of each heparinase 1, 2, and 3. One
skilled in the art can readily adapt these exemplary conditions to
different amounts of substrate and enzyme.

[0101]According to the method of the invention, the various
polysaccharides and oligosaccharides present in a depolymerized sample
are separated and quantified using methods known to those skilled in the
art. For example, polysaccharides and oligosaccharides comprising
glycoserine and its oxidized derivatives may be separated by HPLC using
anion-exchange chromatography, for example, with a stationary phase
grafted with quaternary ammonium derivatives such as --NMe3.sup.+.
The chromatography column may be filled with any of a variety of
available resins, for example, of the Spherisorb SAX type with particle
sizes in the range for example of 5 to 10 um.

[0102]The apparatus used may be any chromatograph that allows the
formation of an elution gradient and that can utilize a UV detector. In
one embodiment of the invention, the UV detector may be a diode array
that can measure UV spectra for the constituents on at least two
different wavelengths and record signals resulting from the difference
between the absorbances at these different wavelengths. This allows
specific detection of acetylated oligosaccharides. To allow this type of
detection, mobile phases that are transparent to UV light up to 200 nm
are preferable. Exemplary mobile phases may be based on perchlorate,
methanesulfonate, or phosphate salts. And the pH of the mobile phase for
the separation may be from about 2.0 to about 6.5. In one particular
embodiment, the pH of the mobile phase is about 3. As nonacetylated
polysaccharides all have, at a given pH, quite a similar UV spectrum, it
is possible to selectively detect acetylated sugars by taking, as the
signal, the difference between the absorbance at two wavelengths chosen
such that the absorptivity of the nonacetylated saccharides is cancelled
out.

[0103]Quantification of polysaccharides and oligosaccharides comprising
glycoserine and its oxidized derivatives may be carried out using methods
for external or internal calibration. In one embodiment of the invention,
the internal standard used is 2-naphthol-3,6-disulfonic acid. In this
embodiment, the aqueous solution of heparin to be assayed contains about
0.15 g/l of 2-naphthol-3,6-disulfonic acid. The depolymerized sample may
be assayed by chromatography according to the method described in patent
FR 0211724 filed Sep. 23, 2002, which is incorporated by reference
herein, and illustrated below for its specific application to the method
for assaying for glycoserine content.

[0112]A chromatogram representative of the result of analyzing a
depolymerized heparin material according to these exemplary conditions is
shown in FIG. 2.

[0113]In particular, a subject of the invention is the method of analysis
as defined above, wherein the presence of tetrasaccharide characteristic
of the linking domain (the glycoserine) is sought. This tetrasaccharide
is:

[0114]Another aspect of the invention is the method of detection utilized
during chromatographic separation. In particular, a method has been
developed in order to increase the specificity of the UV detection. As
shown in FIG. 3, 202 nm and 240 nm will be chosen as detection and
reference wavelength and the 202-240 nm signal will be noted. A suitable
detector for this technique is the DAD 1100 detector from the company
Agilent Technologies. In this case, a double detection will be carried
out, firstly, at 234 nm and, secondly at 202-240 nm.

[0115]A subject of the present invention is also a method of analysis as
defined above, using separation by anion-exchange chromatography, wherein
the detection method makes it possible to selectively detect acetylated
sugars.

[0116]A subject of the invention is also a method of analysis as defined
above, using separation by exchange chromatography, wherein the selective
detection of the acetylated sugars is carried out by taking, as the
signal, the difference between the absorbance at two wavelengths chosen
such that the absorptivity of the nonacetylated saccharides is cancelled
out.

[0117]Preparation for Calorimetric Analysis of the Sample

[0118]A solution in water may be prepared so that the concentration is
adjusted to 100 mg/ml, and the solution obtained is then filtered through
a membrane filter with a porosity of 0.22 μm. The value of the
absorbance at 400 nm is then measured. This measurement corresponds to
the initial value for the study. The solution is distributed into seven
bottles at a rate of approximately 5 ml per bottle. The samples are then
set by being placed in an oven at 45° C. Each week, a bottle is
taken out of the oven and the value of the absorbance at 400 nm is
measured at 20° C. After 6 weeks, the suitability of the sample
can be determined.

[0141]Thus, the method of the present invention includes a method for
pre-treating the sample before it is analyzed and the chromatography
process used for determining the presence or absence of the
tetrasaccharide characteristic of the linking domain.

EXAMPLES

[0142]In the following examples the abbreviation "NI" means internal
normalization. Examples 6-11 demonstrate the influence of temperature and
potassium permanganate concentration on the elimination of glycoserine
residues from heparin.

Example 1

Preparation of "Upgraded" Heparin

[0143]This process increases the anti-Xa activity of a crude heparin
preparation and may be employed before using a heparin preparation in the
process to remove glycoserine residues. The "upgraded" heparin prepared
in this example was used in examples 2-10. And the initial anti-Xa
activity of the crude heparin preparation used in this example is 150
IU/mg.

[0144]Five hundred and twenty milliliters of water and 112.5 g of NaCl
(20% w/v) were introduced into a 2 L reactor. After dissolution, 60.0 g
of crude heparin was added to the solution. After mechanical agitation
for 30 minutes, the precipitate in suspension was filtered through a
number 3 sintered glass funnel packed with 33.9 g of clarcel. The
sintered glass filter was then rinsed with 120 ml of 20% (w/v) NaCl
solution. The filtrate obtained (704 ml) was loaded into a 2 L reactor
and methanol (388 ml) was rapidly poured in, in the presence of
mechanical stirring. After stirring for approximately 2 hours at a
temperature of about 20° C., the suspension was left to sediment
overnight. The supernatant (804 ml) was removed and discarded and the
sedimented precipitate was taken up in a 20% NaCl solution (73.5 g of
NaCl in 368 ml of water). After stirring for 30 minutes, 385 ml of
methanol were rapidly added. After stirring for approximately 1 hour at a
temperature of about 20° C., the suspension was left to sediment
for approximately 12 hours. The supernatant (840 nil) was then removed
and discarded and methanol (400 ml) was added to the sedimented
precipitate. Again, after stirring for approximately 1 hour, the
suspension was left to sediment for approximately 12 hours and the
supernatant (430 ml) was removed and discarded. Methanol (400 ml) was
added to the sedimented precipitate. After stirring this solution for 1
hour, the suspension was left to sediment for approximately 12 hours.
After sedimentation, the suspended precipitate was filtered through a
number 3 sintered glass funnel. The cake obtained was washed twice with
400 ml of methanol. The wet solid was filter-dried and then dried under
reduced pressure (6 kPa), at a temperature of about 40° C. After
drying, 43.7 g of "upgraded" heparin was obtained. The yield was 73%.

[0145]Glycoserine residues are not affected by these treatments and the
percentage of glycoserine in the sample (NI %) was 2.4.

[0146]Twelve grams of "upgraded" heparin obtained as described in example
1 and 120 ml of distilled water were introduced into a 250 ml Erlenmeyer
flask. The temperature of the mixture was adjusted to 40° C. with
magnetic stirring. The pH of the mixture was adjusted to 8.7±0.3 by
adding 1 N sodium hydroxide. The reaction medium was heated to 80°
C. and 0.96 g of solid KMnO4 were added. After stirring for 15 minutes,
the mixture was left to flocculate for 1 hour at 80° C. Carcel
(1.2 g) was then added. After stirring for 15 minutes, the precipitate
was collected by filtration through a number 3 sintered glass filter
packed with 15 g of clarcel. The precipitate was washed with 25 ml of
water at 60° C. and then with 25 ml of water at 20° C.
Then, the filtrate (180 ml) was placed in a 250 ml Erlenmeyer flask and
1.8 g NaCl was added to the filtrate in order to obtain a 1% solution.
The pH was adjusted to approximately 11.2±0.3 by adding 1 N sodium
hydroxide. The reaction medium was heated at 45° C. for 2 hours,
and was then left to stir slowly at about 20° C. for approximately
12 hours. This solution was filtered through a number 3 sintered glass
funnel and the funnel was washed with 15 ml of 20% (w/v) NaCl solution.
The filtrate was placed in a 250 ml Erlenmeyer flask, and 1.2 ml of a 30%
aqueous hydrogen peroxide solution was added. The reaction medium was
left to stir slowly at about 20° C. for approximately 12 hours.
The pH was adjusted to 6.2±0.3 by adding a 6N HCl solution and the
NaCl concentration was adjusted to 3%. This solution was filtered through
a membrane with a porosity of 1 μm and the membrane was washed with 5
ml of water. The filtrate (204 ml) gathered from this filtration was
placed in a 500 ml Erlenmeyer flask and methanol (163 ml) was added
rapidly in the presence of magnetic stirring. After vigorous stirring for
30 minutes, the suspension was left to sediment for approximately 12
hours. The supernatant (295 ml) was then removed and discarded and
methanol (70 ml) was added to the sedimented precipitate. After stirring
for approximately 30 minutes, the suspension was left to sediment for 3
hours. Then supernatant (80 ml) was removed and discarded and methanol
(60 ml) was added to the sedimented precipitate. After stirring for 30
minutes, the suspension was left to sediment for approximately 12 hours.
The precipitate in suspension was filtered through a number 3 sintered
glass funnel. The cake obtained from this filtration was washed with two
portions of 25 ml of methanol. The wet solid was filter-dried and then
dried under reduced pressure (6 kPa), at a temperature of about
40° C. After drying, 10.33 g of "purified" heparin were obtained.
The yield obtained was 86.1%.

[0147]The composition obtained had the following characteristics:

[0148]NI % Glycoserine=0

[0149]Anti-Xa activity=203.5 IU/mg

Example 3

"Upgraded" Heparin Without Oxidation and With Depyrogenation

[0150]Twelve grams of "upgraded" heparin obtained as described in example
1 and 120 ml of distilled water were introduced into a 250 ml Erlenmeyer
flask. NaCl (1.21 g) was added in order to obtain a 1% solution. The pH
was adjusted to approximately 11.2±0.3 by adding 1 N sodium hydroxide.
The reaction medium was heated at 45° C. for 2 hours, and was then
left to stir slowly at a temperature of about 20° C. for
approximately 12 hours. The solution was filtered through a number 3
sintered glass funnel and the funnel was washed with 10 ml of 20% (w/v)
NaCl solution. The filtrate obtained was placed into a 250 ml Erlenmeyer
flask, and 1.2 ml of a 30% aqueous hydrogen peroxide solution were added.
The reaction medium was stirred slowly at a temperature of about
20° C. for 12 hours. The pH was adjusted to 6.2±0.3 by adding a
6N HCl solution and the NaCl concentration was adjusted to 3%. This
solution was filtered through a membrane with a porosity of 1 μm. The
filtrate (138 ml) was placed in a 250 ml Erlenmeyer flask and methanol
(110 ml) was added rapidly in the presence magnetic stirring. After
stirring for 30 minutes, the suspension was left to sediment for
approximately 12 hours. The supernatant (195 ml) was then removed and
discarded and methanol (55 ml) was added to the sedimented precipitate.
After stirring for approximately 30 minutes, the suspension was left to
sediment for approximately 1 hour. The supernatant (57 ml) was again
removed and discarded. Methanol (55 ml) was added to the sedimented
precipitate. After stirring for 30 minutes, the suspension was left to
sediment for approximately 12 hours. The precipitate was then collected
by filtration through a number 3 sintered glass funnel and the cake
obtained was washed with 2 portions of 25 ml of methanol. The wet solid
was filter-dried and then dried under reduced pressure (6 kPa), at a
temperature of about 40° C. After drying, 11.12 g of "purified"
heparin was obtained. The yield obtained was 92.7%.

[0151]The composition obtained had the following characteristics:

[0152]NI % Glycoserine=2.4

[0153]Anti-Xa activity=203.7 IU/mg

Example 4

"Upgraded" Heparin Without Oxidation and Without Depyrogenation

[0154]12 g of "upgraded" heparin obtained as described in example 1 and
120 ml of distilled water were introduced into a 250 ml Erlenmeyer flask.
The pH was brought to approximately 11.2±0.3 by adding 1 N sodium
hydroxide, and 1.2 ml of an aqueous hydrogen peroxide solution were
added. The reaction medium was left to stir slowly at a temperature in
the region of 20° C. for 12 hours. The pH was brought back to
6.2±0.3 by adding 6N HCl solution and the NaCl titer was adjusted to
3%. This solution was filtered through a membrane with a porosity of 1
μm and was washed with 2 ml of water. The filtrate obtained (133 ml)
was placed in a 500 ml Erlenmeyer flask and methanol (106 ml) was added
rapidly thereto in the presence of magnetic stirring. After stirring for
30 minutes, the suspension was left to sediment until the following day.
The supernatant was then removed and discarded (190 ml) and 50 ml of
methanol were added to the sedimented precipitate. After stirring for
approximately 30 minutes, the suspension was left to sediment for 4
hours. The supernatant was again removed and then discarded (48 ml) and
50 ml of methanol were added to the sedimented precipitate. After
stirring for 30 minutes, the suspension was then left to sediment for
approximately 12 hours. After sedimentation, the precipitate in
suspension was filtered through a number 3 sintered glass funnel. The
cake obtained was then washed with two portions of 25 ml of methanol. The
wet solid was filter-dried and then dried under reduced pressure (6 kPa),
at a temperature in the region of 40° C. After drying, 10.48 g of
"purified" heparin were obtained. The yield obtained was 87.35%.

[0155]The composition obtained had the following characteristics:

[0156]NI % Glycoserine=2.4

[0157]Anti-Xa activity=209.5 IU/mg

Example 5

"Upgraded" Heparin With Oxidation and Without Depyrogenation

[0158]12 g of "upgraded" heparin obtained according to example 1 and 120
ml of distilled water were introduced into a 250 ml Erlenmeyer flask. The
mixture was brought to 40° C. in the presence of magnetic
stirring. The pH was brought to 8.7±0.3 by adding 1 N sodium
hydroxide. The reaction medium was heated to about 80° C. and 0.96
g of solid KMnO4 were added to the reaction medium. After stirring for 15
minutes, the mixture was left to flocculate for 1 hour at 80° C.
1.2 g of clarcel was then added. After stirring for 15 minutes, the
precipitate in suspension was filtered through a number 3 sintered glass
funnel packed with 15 g of clarcel. This was successively washed with 25
ml of water at 60° C. and then with 25 ml of water at 20°
C. The filtrate (178 ml) was placed in a 250 ml Erlenmeyer flask and 1.2
ml of a 30% aqueous hydrogen peroxide solution was added thereto. The
reaction mixture was left to stir slowly at a temperature in the region
of 20° C. for 12 hours. The pH was brought back to 6.2±0.3 by
adding a 6N HCl solution and the NaCl titer was adjusted to 3%. This
solution was then filtered through a membrane with a porosity of 1 μm
and washed with 2 ml of water. The filtrate obtained (192 ml) was placed
in a 500 ml Erlenmeyer flask and 154 ml of methanol were rapidly added
thereto, in the presence of magnetic stirring. After stirring for 30
minutes, the suspension was left to sediment until the following day. The
supernatant was then removed and discarded (283 ml) and 60 ml of methanol
were added to the precipitated sediment. After stirring for approximately
30 minutes, the suspension was left to sediment for 4 hours. The
supernatant was again removed and discarded (58 ml) and another 60 ml of
methanol were added to the sedimented precipitate. After stirring for 30
minutes, the suspension was left to sediment for approximately 12 hours.
The precipitate in suspension was filtered through a number 3 sintered
glass funnel. The cake obtained was then washed with two portions of 25
ml of methanol. The wet solid was filter-dried and then dried under
reduced pressure (6 kPa), at a temperature in the region of 40° C.
After drying, 10.31 g of "purified" heparin were obtained. The yield
obtained was 85.9%.

[0159]The composition obtained had the following characteristics:

[0160]NI % Glycoserine=0

[0161]Anti-Xa activity=210.3 IU/mg

Example 6

"Upgraded" Heparin Oxidized in 8% KMnO4 at 80° C.

[0162]10 g of "upgraded" heparin obtained according to example 1 and 100
ml of distilled water were introduced into a 250 ml Erlenmeyer flask. The
mixture was brought to 40° C. in the presence of magnetic
stirring. The pH was brought to 8.7±0.3 by adding 1 N sodium
hydroxide. The reaction medium was heated to 80° C. and 0.80 g of
solid KMnO4 were added to it. After stirring for 15 minutes, the mixture
was left to flocculate for 1 hour at 80° C. 1.0 g of clarcel was
then added. After stirring for 15 minutes the precipitate in suspension
was filtered through a number 3 sintered glass funnel packed with 15 g of
clarcel. This was successively washed with 25 ml of water at 60°
C. and then with 25 ml of water at 20° C. The filtrate obtained
(152 ml) was placed in a 250 ml Erlenmeyer flask and 1.5 g of NaCl were
added thereto in order to obtain a 1% solution. The pH was brought to
approximately 11±0.3 by adding 1 N sodium hydroxide. The reaction
medium was heated to approximately 45° C. for 2 hours, and was
then left to stir slowly at a temperature in the region of 20° C.
for 12 hours. The solution was filtered through a number 3 sintered glass
funnel. The filtrate obtained was introduced into a 250 ml Erlenmeyer
flask, and 1.0 ml of a 30% aqueous hydrogen peroxide solution were added
thereto. The reaction medium was left to stir slowly at a temperature in
the region of 20° C. for 12 hours. The pH was brought back to
6.2±0.3 with a 6N HCl solution and the NaCl titer was adjusted to 3%.
This solution was filtered through a membrane with a porosity of 1 μm
and half the filtrate (70 ml) was placed in a 250 ml Erlenmeyer flask and
56 ml of methanol were added rapidly thereto, in the presence of magnetic
stirring. After stirring for 30 minutes, the mixture was left to
sediment. The supernatant was removed and then discarded (118 ml) and 60
ml of methanol were added to the sedimented precipitate. After stirring
for 30 minutes, the suspension was left to sediment for approximately 12
hours. The supernatant was then removed and discarded (54 ml) and 50 ml
of Methanol were added to the sedimented precipitate. After stirring for
approximately 30 minutes, the suspension was left to sediment for
approximately 12 hours. The precipitate in suspension was filtered
through a number 3 sintered glass funnel. The cake obtained was then
washed with two portions of 25 ml of methanol. The wet solid was
filter-dried and then dried under reduced pressure (6 kPa), at a
temperature in the region of 40° C. After drying, 2.80 g of
"purified" heparin were obtained. The yield obtained was 63.3%.

[0163]The composition obtained had the following characteristics:

[0164]NI % Glycoserine=0.0

Example 7

"Upgraded" Heparin Oxidized in 4% KMnO4 at 80° C.

[0165]10 g of "upgraded" heparin obtained according to example 1 and 100
ml of distilled water were introduced into a 250 ml Erlenmeyer flask. The
mixture was brought to 40° C. in the presence of magnetic
stirring. The pH was brought to 8.7±0.3 by adding 1 N sodium
hydroxide. The reaction medium was heated to 80° C. and 0.40 g of
solid KMnO4 were added thereto. After stirring for 15 minutes, the
mixture was left to flocculate for 1 hour at 80° C. 1.0 g of
clarcel was added thereto. After stirring for 15 minutes, the precipitate
in suspension was filtered through a number 3 sintered glass funnel
packed with 15 g of clarcel. This was successively washed with 25 ml of
water at 60° C. and then with 25 ml of water at 20° C. The
filtrate obtained (158 ml) was placed in a 250 ml Erlenmeyer flask and
1.6 g of NaCl were added thereto in order to obtain a 1% solution. The pH
was brought to approximately 11±0.3 by adding 1 N sodium hydroxide.
The reaction medium was heated to 45° C. for 2 hours, and was then
left to stir slowly at a temperature in the region of 20° C. for
12 hours. The solution was filtered through a number 3 sintered glass
funnel. The filtrate was again introduced into a 250 ml Erlenmeyer flask,
and 1.0 ml of a 30% aqueous hydrogen peroxide solution was added. The
reaction medium was left to stir slowly at a temperature in the region of
20° C. for 12 hours. The pH was brought back to 6.2±0.3 with a
6N HCl solution and the NaCl titer was adjusted to 3%. This solution was
filtered through a membrane with a porosity of 1 μm. The filtrate (152
ml) was placed in a 500 ml Erlenmeyer flask and 122 ml of methanol were
added rapidly thereto, in the presence of magnetic stirring. The
supernatant was removed and discarded (251 ml) and 100 ml of methanol
were added to the sedimented precipitate. After stirring for 30 minutes,
the suspension was left to sediment for approximately 12 hours. The
supernatant was then removed and discarded (97 ml) and 100 ml of methanol
were added to the sedimented precipitate. After stirring for
approximately 30 minutes, the suspension was left to sediment for
approximately 12 hours. The precipitate in suspension was filtered
through a number 3 sintered glass funnel. The cake obtained was then
washed with two portions of 30 ml of methanol. The wet solid was
filter-dried and then dried under reduced pressure (6 kPa), at a
temperature in the region of 40° C. After drying, 6.4 g of
"purified" heparin were obtained. The yield obtained was 71%.

[0166]The composition obtained had the following characteristics:

[0167]NI % Glycoserine=0.0

Example 8

"Upgraded" Heparin Oxidized in 8% of KMnO4 at 40° C.

[0168]10 g of "upgraded" heparin obtained according to example 1 and 100
ml of distilled water were introduced into a 250 ml Erlenmeyer flask. The
mixture was brought to 40° C. in the presence of magnetic
stirring. The pH was brought to 8.7±0.3 by adding 1 N sodium
hydroxide. The reaction medium was then heated to 40° C. and 0.80
g of solid KMnO4 were added thereto. After stirring for 15 minutes, the
mixture was left to flocculate for 1 hour at 80° C. 1.0 g of
clarcel was added thereto. After stirring for 15 minutes, the precipitate
in suspension was filtered through a number 3 sintered glass funnel
packed with 15 g of clarcel. This was sequentially rinsed with 25 ml of
water at 60° C. and then 25 ml of water at 20° C. The
filtrate obtained (142 ml) was placed in a 250 ml Erlenmeyer flask and
1.42 g of NaCl were added in order to obtain a 1% solution. The pH was
brought to approximately 11±0.3 by adding 1 N sodium hydroxide. The
reaction medium was heated at 45° C. for 2 hours, and then left to
stir slowly at a temperature in the region of 20° C. for 12 hours.
The solution was then filtered through a number 3 sintered glass funnel.
The filtrate was again introduced into a 250 ml Erlenmeyer flask, and 1.0
ml of a 30% aqueous hydrogen peroxide solution was added. The reaction
medium was left to stir slowly at a temperature in the region of
20° C. for 12 hours. The pH was brought back to 6.2±0.3 with a
6N HCl solution and the NaCl titer was adjusted to 3%. This solution was
filtered through a membrane with a porosity of 1 μm and half the
filtrate (80 ml) was placed in a 250 ml Erlenmeyer flask. 64 ml of
methanol were added rapidly thereto, in the presence of magnetic
stirring. After stirring for approximately 30 minutes, the suspension was
left to sediment. The supernatant was removed and then discarded (131 ml)
and 60 ml of methanol were added to the sedimented precipitate. After
stirring for approximately 30 minutes, the suspension was left to
sediment for approximately 12 hours. The supernatant was then removed and
discarded (52 ml) and 50 ml of methanol were added to the sedimented
precipitate. The suspension was filtered through a number 3 sintered
glass funnel. The cake obtained was then washed with two portions of 25
ml of methanol. The wet solid was filter-dried and then dried under
reduced pressure (6 kPa), at a temperature in the region of 40° C.
After drying, 2.3 g of "purified" heparin were obtained. The yield
obtained was 54%.

[0169]The composition obtained had the following characteristics:

[0170]NI % Glycoserine=0.0

Example 9

"Upgraded" Heparin Oxidized With 8% KMnO4 at 60° C.

[0171]10 g of "upgraded" heparin obtained according to example 1 and 100
ml of distilled water were introduced into a 250 ml Erlenmeyer flask. The
mixture was brought to 40° C. with magnetic stirring. The pH was
brought to 8.7±0.3 by adding 1 N sodium hydroxide. The reaction medium
was heated to 60° C. and 0.80 g of solid KMnO4 were added thereto.
After stirring for 15 minutes, the mixture was left to flocculate for 1
hour at 80° C. 1.0 g of clarcel was then added. After stirring for
15 minutes, the precipitate in suspension was filtered through a sintered
glass funnel 3 packed with 15 g of clarcel. This was then rinsed with 25
ml of water at 60° C. and then 25 ml of water at 20° C. The
filtrate obtained (150 ml) was placed in a 250 ml Erlenmeyer flask. 1.5 g
of NaCl were added in order to obtain a 1% solution. The pH was brought
to approximately 11±0.3 by adding 1 N sodium hydroxide. The reaction
medium was heated at 45° C. for 2 hours, and was then left to stir
slowly at a temperature in the region of 20° C. for 12 hours. The
solution was filtered through a number 3 sintered glass funnel. The
filtrate was again introduced into a 250 ml Erlenmeyer flask, and 1.0 ml
of a 30% aqueous oxygen peroxide solution was added. The reaction medium
was left to stir slowly at a temperature in the region of 20° C.
for 12 hours. The pH was brought back to 6.2±0.3 with a 6N HCl
solution and the NaCl titer was adjusted to 3%. This solution was
filtered through a membrane with a porosity of 1 μm and half the
filtrate (70 ml) was placed in a 250 ml Erlenmeyer flask. 56 ml of
methanol were added rapidly thereto, in the presence of magnetic
stirring. The supernatant was then removed and discarded (118 ml) and 60
ml of methanol were added to the sedimented precipitate. After stirring
for approximately 30 minutes, the mixture was left to sediment for
approximately 12 hours. The supernatant was then removed and discarded
(54 ml) and 50 ml of methanol were added to the sedimented precipitate.
The suspension was filtered through a number 3 sintered glass funnel. The
cake obtained was then washed with two portions of 25 ml of methanol. The
wet solid was filter-dried and then dried under reduced pressure (6 kPa),
at a temperature in the region of 40° C. After drying, 3.12 g of
"purified" heparin were obtained. The yield obtained was 68%.

[0172]The composition obtained had the following characteristics:

[0173]NI % Glycoserine=0.0

Example 10

"Upgraded" Heparin Oxidized in 2% KMnO4 at 80° C.

[0174]10 g of "upgraded" heparin obtained above and 100 ml of distilled
water were introduced into a 250 ml Erlenmeyer flask. The mixture was
brought to 40° C. in the presence of magnetic stirring. The pH was
brought to 8.7±0.3 by adding 1 N sodium hydroxide. The reaction medium
was heated to 80° C. and 0.16 g of solid KMnO4 were added thereto.
After stirring for 15 minutes, the mixture was left to flocculate for 1
hour at 80° C. 1.0 g of clarcel was then added. After stirring for
15 minutes, the precipitate in suspension was filtered through a number 3
sintered glass funnel packed with 15 g of clarcel. This was then rinsed
with 25 ml of water at 40° C. and then 25 ml of water at
20° C. The filtrate (114 ml) was placed in a 250 ml Erlenmeyer
flask. 1.14 g of NaCl were added in order to obtain a 1% solution. The pH
was brought to approximately 11±0.3 by adding 1 N sodium hydroxide.
The reaction medium was heated at 45° C. for 2 hours, and was then
left to stir slowly at a temperature in the region of 20° C. for
12 hours. The solution was filtered through a number 3 sintered glass
funnel. The filtrate was again introduced into a 250 ml Erlenmeyer flask,
and 0.8 ml of a 30% aqueous hydrogen peroxide solution was added. The
reaction medium was left to stir slowly at a temperature in the region of
20° C. for 12 hours. The pH was brought back to 6.2±0.3 with a
6N HCl solution and the NaCl titer was adjusted to 3%. This solution was
filtered through a membrane with a porosity of 1 μm and half the
filtrate (75 ml) was placed in a 250 ml Erlenmeyer flask. 60 ml of
methanol were added rapidly, in the presence of magnetic stirring. After
stirring for approximately 30 minutes, the mixture was left to sediment.
The supernatant was removed and then discarded (120 ml) and 50 ml of
methanol are added to the sedimented precipitate. After stirring for
approximately 30 minutes, the mixture was left to sediment. The
supernatant was then removed and discarded (38 ml) and 40 ml of methanol
were added to the sedimented precipitate. After stirring for
approximately 30 minutes, the suspension was left to sediment. The
supernatant was then removed and discarded (27 ml) and 30 ml of methanol
were added to the sedimented precipitate. The suspension was filtered
through a number 3 sintered glass funnel. The cake obtained was then
washed with two portions of 25 ml of methanol. The wet solid was
filter-dried and then dried under reduced pressure (6 kPa) at a
temperature in the region of 40° C. After drying, 2.61 g of
"purified" heparin were obtained. The yield obtained was 72%.

[0175]The composition obtained had the following characteristics:

[0176]NI % Glycoserine=0.8

Example 11

"Upgraded" Heparin Oxidized in 2% KMnO4 at 60° C.

[0177]10 g of "upgraded" heparin obtained according to example 1 and 100
ml of distilled water were introduced into a 250 ml Erlenmeyer flask. The
mixture was brought to 40° C. in the presence of magnetic
stirring. The pH was brought to 8.7±0.3 by adding 1 N sodium
hydroxide. The reaction medium was heated to 60° C. and 0.16 g of
solid KMnO4 were added thereto. After stirring for 15 minutes, the
mixture was left to flocculate for 1 hour at 60° C. 1.0 g of
clarcel was then added. After stirring for 15 minutes, the precipitate in
suspension was filtered through a number 3 sintered glass funnel packed
with 15 g of clarcel. This was then rinsed with two portions of 20 ml of
water at 40° C. The filtrate (129 ml) was placed in a 250 ml
Erlenmeyer flask. 1.29 g of NaCl were added thereto in order to obtain a
1% solution. The pH was brought to approximately 11±0.3 by adding 1 N
sodium hydroxide. The reaction medium was heated at 45° C. for 2
hours, and was then left to stir slowly at a temperature in the region of
20° C. for 12 hours. The solution was filtered through a number-3
sintered glass funnel. The filtrate was again introduced into a 250 ml
Erlenmeyer flask, and 0.8 ml of a 30% aqueous hydrogen peroxide solution
was added. The reaction medium was left to stir slowly at a temperature
in the region of 20° C. for 12 hours. The pH was brought back to
6.2±0.3 with a 6N HCl solution and the NaCl titer was adjusted to 3%.
This solution was filtered through a membrane with a porosity of 1 μm
and half the filtrate (75 ml) was placed in a 250 ml Erlenmeyer flask. 60
ml of methanol were added rapidly thereto, in the presence of magnetic
stirring. The supernatant was removed and then discarded (120 ml) and 50
ml of methanol were added to the sedimented precipitate. After stirring
for approximately 30 minutes, the mixture was left to sediment. The
supernatant was then removed and then discarded (32 ml) and 30 ml of
methanol were added to the sedimented precipitate. After stirring for
approximately 30 minutes, the mixture was left to sediment. The
supernatant was then removed and discarded (26 ml) and 30 ml of methanol
were added to the sedimented precipitate. The suspension is filtered
through a number 3 sintered glass funnel. The cake obtained was then
washed with two times 25 ml of methanol. The wet solid was filter-dried
and then dried under reduced pressure (6 kPa), at a temperature in the
region of 40° C. After drying, 2.24 g of "purified" heparin were
obtained. The yield obtained was 62%.